Biological sex affects vaccine efficacy and protection against influenza in mice

Abstract

Biological sex affects adaptive immune responses, which could impact influenza infection and vaccine efficacy. Infection of mice with 2009 H1N1 induced antibody responses, CD4⁺ T cell and CD8⁺ T cell memory responses that were greater in females than males; both sexes, however, were equally protected against secondary challenge with an H1N1 drift variant virus. To test whether greater antibody in females is sufficient for protection against influenza, males and females were immunized with an inactivated H1N1 vaccine that induced predominantly antibody-mediated immunity. Following vaccination, females had greater antibody responses and protection against challenge with an H1N1 drift variant virus than males. Antibody derived from vaccinated females was better at protecting both naïve males and females than antibody from males, and this protection was associated with increased antibody specificity and avidity to the H1N1 virus. The expression of Tlr7 was greater in B cells from vaccinated females than males and was associated with reduced DNA methylation in the Tlr7 promoter region, higher neutralizing antibody, class switch recombination, and antibody avidity in females. Deletion of Tlr7 reduced sex differences in vaccine-induced antibody responses and protection following challenge and had a greater impact on responses in females than males. Taken together, these data illustrate that greater TLR7 activation and antibody production in females improves the efficacy of vaccination against influenza.

Keywords: CD8+ T cell memory; DNA methylation; antibody secreting cells; isotope switching; toll-like receptor 7.

Fig. 1.

Females have greater antibody responses and B cell activation in response to influenza A virus infection. Adult male and female mice were inoculated intranasally with 10 TCID₅₀ (i.e., the tissue culture infectious dose that causes cytopathic effects in 50% of cells) units of 2009 H1N1 virus, serum was collected to measure anti-2009 H1N1 IgG titers (A; n = 10/sex/time point), and BAL fluid was collected to analyze anti-2009 H1N1 IgA titers (B; n = 9–10/sex/time point). At 21 dpi, anti-2009 H1N1 IgA antibody-secreting (ASC) B cells (n = 6 pools of 3/sex) were quantified in the lungs (C) and CD4⁺ T cells (D), T-follicular helper cells (E), and germinal center B cells (F) were analyzed in the lung-draining mLNs by flow cytometry (n = 13–14/sex); M = males, F = females). At 21 dpi, mLNs were isolated and frozen sections were stained for germinal centers by immunofluorescence (G). The ratio of each germinal center area to the area of the lymph node section was calculated using ImageJ (n = 3/sex) (H). Data represent means ± SEM from two to three independent experiments and significant differences between males and females are represented by asterisks (*).

Fig. 2.

Influenza A virus infection induces greater numbers CD8⁺ T memory cells in females than males. Male and female mice were inoculated intranasally with 10 TCID₅₀ (i.e., the tissue culture infectious dose that causes cytopathic effects in 50% of cells) units of 2009 H1N1 at day 0 and on day 42 postinfection, half of the mice were either killed to measure virus-specific CD8⁺ T cells or challenged with 10⁵ TCID₅₀ units of 2009dv virus and killed 6 d postchallenge to quantify memory responses. Total numbers of live CD8⁺ tetramer⁺ cells (A), CD8⁺ memory cells (B), CD8⁺ central memory (TCM) cells (C), and CD8⁺ TRM cells (D) were quantified; M = males, F = females. Morbidity following challenge was determined by monitoring changes in body mass (numbers represent the number of animals that survived the challenge out of the total in each group) (E), and virus titers were quantified in the lungs at 3 d postchallenge (F) in vaccinated (Live Vacc) and unvaccinated (Unvacc) mice. Data represent means ± SEM from two independent experiments (n = 8–10/sex) and significant differences between males and females and treatment groups are represented by asterisks (*).

Fig. 3.

Inactivated influenza virus vaccination induces greater antibody responses and better protection in females. Adult male and female mice were vaccinated with inactivated 2009 H1N1 on day 0, boosted on day 21, and challenged with 10⁵ TCID₅₀ (i.e., the tissue culture infectious dose that causes cytopathic effects in 50% of cells) units of 2009dv virus on day 42. Anti-2009 H1N1 IgG (A) and neutralizing antibody (nAb; B) titers in serum were determined. Morbidity was determined by monitoring changes in body mass (C), and virus titers were quantified in the lungs after challenge (D). At 6 wk postvaccination, serum was pooled by sex and intraperitoneally injected into naïve mice of both sexes. Male and female control animals received serum from naïve males and females. Three hours later, mice were bled and challenged with the 2009dv virus, and 3 d later lung virus titers were quantified (E). Lung virus titers were correlated with IgG antibody titers (F). Data represent means ± SEM from two independent experiments (n = 7–10/sex) and significant differences between groups are represented by asterisks (*).

Fig. 4.

Inactivated influenza virus-vaccinated females generate greater quality antibodies than vaccinated males. Adult male and female mice were vaccinated with inactivated 2009 H1N1 on day 0, boosted on day 21, and antibody responses were measured on day 28. Anti-2009 H1N1 IgM (A), IgG1 (B), IgG2c (C), IgG1:IgG2c ratio (D), and IgG avidity index (E) were measured in serum; M = males, F = females. Data represent means ± SEM from two independent experiments (n = 9–10/sex) and significant differences between males and females are represented by asterisks (*).

Fig. 5.

Tlr7 expression is elevated in female-derived B cells and is necessary for greater antibody titers and protection in vaccinated females than males. Adult male and female mice were vaccinated with inactivated 2009 H1N1 or vehicle on day 0, boosted on day 21, and splenic B cells were isolated on day 28. RNA was extracted from B cells to evaluate the relative gene expression of Tlr7 (A) and Tlr8 (B). Using DNA isolated from B cells, CpG site-specific DNA methylation of the 5′ regulatory region for Tlr7 was assayed. Results are represented as the change in the percentage of DNA methylation on Tlr7 in vaccinated relative to unvaccinated males or females (C; n = 5–9/sex). To evaluate the contribution of Tlr7 to antibody somatic hypermutation and class switch recombination, adult male and female wild-type and Tlr7 knockout mice were vaccinated with inactivated 2009 H1N1 on day 0, boosted on day 21, and antibody responses were measured on day 28. Anti-2009 H1N1 neutralizing antibody (D), IgG (E), IgG1 (F), IgG2c (G), and IgG avidity (H) responses were measured in serum. To evaluate protection following challenge, male and female wild-type (WT) and Tlr7 knockout (KO) mice were vaccinated and boosted with inactivated 2009 H1N1 and challenged with 10⁵ TCID₅₀ (i.e., the tissue culture infectious dose that causes cytopathic effects in 50% of cells) units of 2009dv virus on day 42. Virus titers were measured in the lungs after challenge (I). Data represent means ± SEM from two to three independent experiments (n = 8–14/sex/genotype) and significant differences between groups are represented by asterisks (*).